1. Field of the Invention
This invention relates to an optical detection method and an optical detection apparatus for individually identifying fine particles such as cells or micro beads. More specifically, the present invention relates to an optical detection method and an optical detection apparatus wherein the type of a fine particle is identified from fluorescent light or scattered light generated from the fine particle when a laser beam of a particular wavelength is irradiated upon the fine particle.
2. Description of the Related Art
Generally, where it is intended to identify a living body-related small particle such as a cell, a microorganism or a ribosome, an optical detection method which uses a flow cytometry or flow cytometer is used as disclosed, for example, in Hiromitsu NAKAUCHI, “Cellular Engineering Separate Volume, Experiment Protocol Series, Master Flow cytometry”, Shujunsha Co. Ltd., the second edition, Aug. 31, 2006 (hereinafter referred to as Non-Patent Document 1). The flow cytometry is a method of irradiating a laser beam of a particular wavelength on one of fine particles which are successively fed in a row in a flow path and detecting fluorescent light or scattered light generated from the fine particle to discriminate a plurality of fine particles one by one.
In particular, in the flow cell, a laminar flow is formed from sample liquid which includes fine particles of an object of detection and sheath liquid which flows around the sample liquid. Further, a very small pressure difference is applied between the sample liquid and the sheath liquid to arrange a plurality of fine particles included in the sample liquid into a row. If, in this state, a laser beam is irradiated upon the flow cell, then the fine particles pass one by one crossing the laser beam. At this time, fluorescent light and/or scattered light excited by the laser light and generated from each fine particle is detected using an electro-optical detector.
Then, the detected light is converted into an electric signal and into a numerical value to carry out statistic analysis thereby to decide the type, size, structure and so forth of each fine particle. It is to be noted that, if each of fine particles of an object of detection is qualified with a plurality of fluorescent dyes, then even if an optical filter is used to separate the detected light for individual wavelength bands, fluorescent light from a fluorescent dye which is not a target fluorescent dye sometimes leaks into a detector different from the detector for the target fluorescent dye. Therefore, in a detection method in related art, in order to obtain only data from object fluorescent light, fluorescent light correction of subtracting an overlap of fluorescent light is usually carried out.
Further, with the flow cytometry, since the type, size, structure and so forth of fine particles can be identified individually and a plurality of parameters can be analyzed simultaneously, even where sample liquid includes a plurality of kinds of fine particles, only necessary fine particles can be dispensed rapidly and with certainty.
Also a flow cytometer which guides, in order to make it possible to detect a plurality of fluorescent lights generated from a fine particle qualified with a plurality of fluorescent dyes, laser beams of wavelengths different from each other along the same incoming light path so as to be irradiated upon the fine particle has been proposed in the past. A flow cytometer of the type is disclosed in Japanese Patent Laid-Open No. 2007-046947 (hereinafter referred to as Patent Document 1). FIG. 12 schematically shows a configuration of the flow cytometer in related art disclosed in Patent Document 1. Referring to FIG. 12, the flow cytometer 101 includes a flow system 102 for arraying cells qualified with fluorescent dyes into one array in the flow cell, and an optical system 103 for irradiating a plurality of laser beams of different wavelengths from each other upon the cells to detect detection object light such as scattered light and fluorescent light. The flow cytometer 101 further includes a signal processing apparatus 104 for controlling and processing an electric signal relating to the scattered light and the fluorescent light outputted from the optical system 103.
In the related-art flow cytometer 101, a plurality of laser beams having different wavelengths from each other are irradiated in a predetermined period and in different phases from each other from light sources 106a, 106b and 106c. The laser beams are guided to the same incoming light path and condensed on a cell 105 by a light guide member 107. Consequently, even where a plurality of laser beams are irradiated upon a cell labeled with a plurality of fluorescent dyes, one incoming light path can be used. Therefore, a plurality of fluorescent lights emitted from a cell can be detected without setting delay time.
Further, also a method has been proposed wherein, in order to dispense live cells which can be utilized for regenerative medicine, interplanting and so forth, object cells are separated and dispensed without carrying out fluorescent labeling wherein a probe such as an antibody to which a fluorescent dye is coupled is used. The method is disclosed in Japanese Patent Laid-Open No. 2003-304867 (hereinafter referred to as Patent Document 2). In the method disclosed in Patent Document 2, forwardly scattered light and backwardly scattered light emitted from a cell are detected, and detection values of the cell are displayed as position information on a two-dimensional screen to identify the size and the structure of the cell.